Research Paper Volume : 4 | Issue : 6 | June 2015 • ISSN No 2277 - 8179 Botany Analysis of genetic diversity of Lagenandra KEYWORDS : , variation, ISSR, spp. (Araceae) of Kerala (South ) using cluster analysis, dendrogram ISSR Markers

Malabar Botanical Garden & Institute for Sciences, Calicut – 14, Prakashkumar R. Kerala, India

Anoop K. P. Malabar Botanical Garden & Institute for Plant Sciences, Calicut – 14, Kerala, India

Sivu A. R. Department of Botany, N.S.S. College, Nilamel, Kerala, India

Ansari R. Malabar Botanical Garden & Institute for Plant Sciences, Calicut – 14, Kerala, India

Pradeep N. S. JNTBGRI, Palode, Thiruvananthapuram, Kerala, India

Madhusoodanan P. V. Malabar Botanical Garden & Institute for Plant Sciences, Calicut – 14, Kerala, India

ABSTRACT The Present study reveals the molecular variations in different species of Lagenandra, an collected from different geographical areas of Kerala State, India. Molecular analysis was carried out using ISSR markers. Out of the 18 primers screened, a total of 66 scorable polymorphic markers were generated. The genetic distance between the population ranged from 0.0016 to 0.0271 and the genetic identity ranged from 0.9732 to 0.9984. The standard deviation of Gene diversity, Shannon's Information index and effective number of alleles are about 0.1468, 0.0719 and 0.0866 respectively. It is evident that that there is distinct genetic variability among Lagenandra species, occurring in Kerala

Introduction: (Mirali & Nabulsi, 2003), ISSR (Wang et al, 2004), AFLP (Rotten- The Lagenandra coming under the angiosperm family Ar- berg & Parker, 2003), and SSR (Rosetto et al, 2004). Inter Simple aceae consist of 15 species, of which six species were reported Sequence Repeats (ISSR) is a molecular marker that has been from Kerala which include Lagenandra keralensis Sivd & Jaleel, widely used in the studies of cultivar identification, genetic Lagenandra meeboldii(Engl.) C.E.C.Fisch., Lagenadra nairii mapping, gene tagging and genetic diversity analysis. Ramam. & Rajan, Lagenandra ovata (L.) Thwaites, Lagenandra toxicaria Dalz.var. toxicaria Hook, Lagenandra toxicaria Dalz. Studies on its level of genetic diversity and pattern of genetic Var. barnesii Fischer. The genus is endemic top Bangladesh, Sri structure of Lagenandra species found in Kerala region were not Lanka and India (Sivadasan et al, 2001). The are peren- undertaken. To conserve a species, it is important to have such nial, all species are ecologically very much alike, and they are basic information. Therefore we decided to examine the genetic usually in and along streams and rivers in forest and planta- variation within and among natural populations of Lagenandra tions but also in irrigation ditches around rice fields. In wet species using ISSR markers. The fact that no DNA sequence in- season they remain submerged and pollination is entomophil- formation is known for the species is one of the main reasons ious. Many species are cultivated for decoration (Cook, 1996). for choosing ISSR technique in this study. On the other hand, the ISSR markers have recently become widely used in population Among the six species, L. ovata (Pop. 1,2,3,4) are comparatively larger studies because they are highly variable, and require less invest- plants. Lagenandra spp. have certain medicinal values; it was evalu- ment in time, money, and labor than other methods. ated for antibacterial activity against gram positive bacteria. The rhizome is used locally in the treatment of kidney disorders, heart Materials and Methods diseases and swelling. L. toxicaria var. toxicaria (Pop. 11,12,15) and Sample collection: Fresh young leaves collected from 15 popu- L.toxicaria var. barnesii(Pop. 13,14) are very much alike, except a few lations of fiveLagenandra species and six varieties collected differences such as in L. toxicaria var. barnesii the petiole is about 3.5 from various localities of Kerala were used in the present study. cm long, the spathe is about 12 cm long opening with wide gap and The accessions (Table: 01) were also maintained in the Aquatic outer surface is greenish pink. While in L. toxicaria var. toxicaria the plant conservatory (Aquagene) of Malabar Botanical Garden petiole is about 9-10 cm long, spathe 6 cm long, open by a narrow Calicut, Kerala. slit and outer surface is greenish purple. These plants are tradition- ally used as water purifying agent because they absorb heavy metals, Table. 01: Species collected for the analysis with Accession so they are often grown in well. Members of the species L. meeboldii code and place of collection (Pop. 5, 7) have some variations in leaf characters but resemble in re- Population Name Accession Collection Localities productive characters. In one population (Pop. 7) the leaves are pur- Code ple colored and the petiole is 13-14 cm long. While in another popu- L. ovata (L.) Thwaites Pop 1 Pariya, Kasarkode lation (Pop. 5) of the same species, leaves are green pale green with white markings and the petiole is 4-5 cm long. L. nairii (Pop 8,9,10) L. ovata (L.) Thwaites Pop 2 Madathara, Kollam are comparatively smaller plants than other species. It is a very rare plant reported only from the Athirapilly waterfalls of Thrissur district L. ovata (L.) Thwaites Pop 3 Munnar, Idukki of Kerala. L. keralensis (Pop. 6) resembles L. meeboldii, but is smaller in size and has differences in the shape of spathe. L. ovata (L.) Thwaites Pop 4 Pala, Kottayam L. meeboldii(Engl.) C.E.C.Fisch Pop 5 Molecular markers have been widely used to characterize pop- Peechi, Thrissur ulation genetic structure of plants. These include allozymes L.keralensisSivd& Jaleel Pop 6 Boothathankettu, and polymerase chain reaction (PCR) based markers like RAPD Ernakulam

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L. meeboldii (Engl.) Pop 7 Nelliyampathy, min. Primers were used (Biogen USA) for the amplification and C.E.C.Fisch Palakkad 25 µl of the amplicon was resolved in 1.2 % agarose gel (Sigma L. nairiiiRamam. &Rajan Pop 8 Athirappally, USA). The gel was stained using ethidium bromide and the DNA Thrissur bands were visualized under UV illuminator. Bands were scored L. nairiiiRamam. &Rajan Pop 9 Athirappally, as present (1) and absent (0). A dendrogram was constructed Thrissur based on the Nei’s mean of distance (Nei, 1972) by UPGMA Athirappally, method. L. nairiiRamam. &Rajan Pop 10 Thrissur L. toxicariaDalz. var. toxicaria Pop 11 Padinjarathara, Results: Wayanad Hook Diversity analysis using Inter-Simple Sequence Repeat (ISSR) L. toxicariaDalz. var. toxicaria Pop 12 Chalakkudy, Thrissur analysis using 18 primers including 15 accession of Lagenandra Hook species from different districts of Kerala revealed the extend of L. toxicariaDalz. var. toxicaria Pop 13 Narikkuni, diversity between the populations. Cluster analysis based on Hook Kozhikode UPGMA reveals 2 well differentiated clusters.In cluster one, L. L. toxicariaDalz. var. barnesii Pop 14 Dhoni forest, ovata, accessions of L. meeboldii and L. keralensis were clus- Fischer Palakkad tered together with 99.51% genetic identity.This cluster includes L. toxicariaDalz. var. barnesii Pop 15 Aruvikkara, two sub clads, one with 4 accessions of L. ovata populations Fischer Thiruvananthapuram grouped together in a single clad with 99.46% similarity. In the other sub clad 2 the 2 accessions of L. meeboldii and L. keralensis ISSR analysis: were clustered with 99.55% genetic identity. 15 populations of five species and six verities of Lagenandra were used for the present study. Total genomic DNA was iso- The second major cluster also include two subclads. The acces- lated from the fresh leaf samples following modified Murray and sions of L.nairii and accessions of L. toxicaria with two varieties Thompson (1980) method using CTAB. ISSR reaction was carried were also included in this major cluster with 99.41% genetic out in 25 µl volume containing 50 ng of Template DNA, 2.5 µl identity. Here one subclad with 3 accessions of L. nairii were of 10x reaction buffers, 50 µM of each dNTP, 20pmol of random grouped with 99.35% genetic identity. The second subclad in- primer, 0.75U Taq DNA Polymerase. Sterile distilled water was cludes two clads one with L. toxicaria Dalz. var. toxicaria and added to the reaction mixture to make the volume to 25 µl. The another with L. toxicaria Dalz. var. barnesii. The former variety PCR reaction was carried following an initial denaturation for grouped with a mean genetic identity of 99.67% and later with 5 min at 95o C, followed by 40 cycles of 1 min. at 95o C, 30 sec. 99.84%. at 50o C and 1 min. at 72o C and a final extension at 72o C for 5

Table No. 02: Nei’s Original Measures of Genetic Identity (above diagonal) and Genetic Distance(below diagonal).

Fig. 01: Dendrogram Based Nei’s (1972) Genetic distance Cluster analysis Lagenandra populations are grouped into 3 clusters (Fig. 01). Cluster I comprises of populations Pop. 1, 2, 3, 4, 5, 6 & 7 col- lected from different localities of Kerala. Cluster II interestingly grouped together the populations Pop. 8, 9 & 10 from Trichur district and the cluster III again comprises populations 11, 12, 13, 14 & 15from different districts of Kerala.

Discussion Knowing the genetic variation is the first step for designing a plant conservation or genetic improvement program. This study demonstrates that ISSR-PCR offered a suitable method for the detection of genetic variability in Lagenandra species. These are semi-aquatic herb found in the marshes and along water cours- es, often growing in semi-evergreen forests.

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The genusLagenandra comprises of two endemic species (L. nar- Acknowledgements irii & L. meboldii) Both medicinal and taxonomic significances The authors are grateful to the Kerala Sate Council for Science, are taken into account for the present diversity analysis using Technology and Environment for the financial assistance and ISSR. The ISSR has been widely used to investigate clonal diver- the authorities of Malabar Botanical Garden Kozhikode for sity and population genetic structure. ISSR data generated in the providing research facilities. We are thankful to the officials of genus Lagenadra collected from different localities of Kerala Kerala Forest Department for providing permission during field showed interesting pattern of genetic diversity existing in the studies. genus. Under cluster I, Pop. 1 and Pop. 2, Pop.3 and Pop.2 and pop. 4 and Pop. 2 showed great genetic diversity between each other. All the populations are coming under the same species Lagenandra ovata and are morphologically very similar but are collected from different climatic areas. Pop.7 and Pop. 5 showed interesting genetic diversity as both these populations are com- ing under the same species Lagenandra meeboldii but have some morphological variations. In population 7 leaves are variegated with pale green markings on dark green leaves and the petiole is short. While in population 5 the leaves are purple cloured and petiole is long (13- 14 cm).

In cluster II- Pop. 10 and Pop. 9 showed high genetic diversity, but both populations belong to same species L. nairii . It is an endemic species reported only from Trissur district. Both the populations are collected from same locality and have no mor- phological dissimilarity.

Under cluster III -Pop.12 and Pop. 11, Pop. 13 and Pop. 11 showed great genetic diversity. But all the populations are under the same species Lagenadra toxicaria var. toxicaria and are mor- phologically so similar but are collected from various geographi- cal areas.

Knowledge of the variation between and within populations of rare and endangered species play a significant role in the formu- lation of appropriate management strategies directed towards their conservation (Milligan et al., 1994). These population ge- netic structure characteristics have significant implication for conservation strategies. Low genetic diversity may reduce the potential of species or population to survive in a changing en- vironment (Ellstrand and Elam, 1993). There is an urgent need to take effective measures to protect these species against fur- ther loss of genetic diversity. This investigation provides valuable information about the nature and pattern of genetic variation exist within different populations of the genus Lagenandra col- lected from different geographical areas.

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